PROJECT SUMMARY/ABSTRACT With more than one million new cases diagnosed yearly, skin cancer accounts for nearly half of all cancers in the U.S. Squamous cell carcinoma (SCC) is a major form of skin cancer with common genetic signatures, but without a clear mechanism of oncogenesis. Developing targeted therapies for SCCs has been challenging as the molecular etiology of the disease paints a heterogeneous picture. An emerging concept is that the synergy or crosstalk between cancer cells and the tumor microenvironment (TME) is critical for tumor initiation and progression. Essential for this cell-stroma crosstalk is the role of membrane-derived, nano-sized extracellular vesicles (EVs) secreted by tumor cells and released into the blood and other bodily fluids carrying their molecular macromolecular cargo to modulate local and distant TME. In addition to mitogenic signaling proteins and mRNAs, EVs carry miRNAs, short non-coding RNAs that repress complementary mRNAs critical for cell cycle regulation and cell proliferation and differentiation. The long-term goal of our research is to understand at the molecular and cellular level, how normal signaling events during development are subverted for pathogenic signaling during SCC progression. During malignant transformation, aberrant expression or localization of the desmosomal cadherin desmoglein 2 (Dsg2) is observed. Dsg2 plays a critical role in normal cell growth and development, which is emphasized by the fact that genetic ablation results in embryonic cell growth defects and lethality in mice. In human, mutations in Dsg2 are associated with cardiomyopathy, respiratory and urinary tract infections, and Alzheimer's disease. Overexpression of Dsg2 also occurs in various other malignancies including prostate, colon and skin, suggesting a more general role for Dsg2 in oncogenesis. The mechanism by which Dsg2 promotes cell proliferation, migration, and hyperproliferative disorders is not fully understood. The goal of this proposal is demonstrate that Dsg2 promotes oncogenesis by enhancing the release of EVs carrying cancer cell-state specific cargos capable of priming keratinocytes and fibroblasts. It is our hypothesis that cell surface presentation of Dsg2 modulates export of EV-associated miRNAs that can serve as biomarkers and key regulators of SCC cancer progression and/or invasion rendering them novel therapeutic targets. Aim 1 will determine the mechanisms by which Dsg2 modulates the biosynthesis of EVs. Aim 2 will study the cellular effects of EV-associated miR146a and miR155. Aim 3 will evaluate the utility of serum EV miRNA levels as prognostic risk factors in SCC. The results obtained here will elucidate novel functions of Dsg2 and shed new light on how Dsg2 can impact cell growth and migration: biological steps critical for proper tissue morphogenesis and tumorigenesis. Upon the successful completion of this work, we will define the mechanisms by which cellular EV export are controlled; how EVs can modulate the stromal environment; and reveal novel miRNAs that can serve as biomarkers for the diagnosis and prognosis of malignant SCCs and other epithelial-derived cancers.